Nostoc sp. PCC 7120 cells that underwent BMAA treatment in diazotrophic conditions. In diazotrophic growth conditions, to survive, cyanobacteria can use only biological nitrogen fixation to obtain nitrogen for life. Note that nitrogen fixation is an energy-consuming process. In total, 1567 different proteins of Nostoc sp. PCC 7120 were identified by using LC-MS/MS spectrometry. Among them, 123 proteins belonging to different functional categories were selected—due to their notable expression differences—for further functional analysis and discussion. The presented proteomic data evidences that BMAA treatment leads to very strong (up to 80%) downregulation of α (NifD) and β (NifK) subunits of molybdenum-iron protein, which is known to be a part of nitrogenase. This enzyme is responsible for catalyzing nitrogen fixation. The genes nifD and nifK are under transcriptional control of a global nitrogen regulator NtcA. In this study, we have found that BMAA impacts in a total of 22 proteins that are under the control of NtcA. Moreover, BMAA downregulates 18 proteins that belong to photosystems I or II and light-harvesting complexes; BMAA treatment under diazotrophic conditions also downregulates five subunits of ATP synthase and enzyme NAD(P)H-quinone oxidoreductase. Therefore, we can conclude that the disbalance in energy and metabolite amounts leads to severe intracellular stress that induces the upregulation of stress-activated proteins, such as starvation-inducible DNA-binding protein, four SOS-response enzymes, and DNA repair enzymes, nine stress-response enzymes, and four proteases. The presented data provide new leads into the ecological impact of BMAA on microalgal communities that can be used in future investigations.Non-proteinogenic neurotoxic amino acid β-N-methylamino-L-alanine (BMAA) is synthesized by cyanobacteria, diatoms, and dinoflagellates, and is known to be a causative agent of human neurodegenerative diseases. Different phytoplankton organisms’ ability to synthesize BMAA could indicate the importance of this molecule in the interactions between microalgae in nature. We were interested in the following: what kinds of mechanisms underline BMAA’s action on cyanobacterial cells in different nitrogen supply conditions. Herein, we present a proteomic analysis of filamentous cyanobacteria  Synechocystis PCC 6803 and Anabaena (Nostoc) PCC 7120 rapidly absorb exogenous BMAA proportionally to the amount of amino acid in the medium ; transketolase  [EC:2.2.1.1]; fructose-bisphosphate aldolase, class II,  [EC:4.1.2.13], and fructose-1,6-bisphosphatase II/sedoheptulose-1,7-bisphosphatase (FBP/SBPase)  [EC:3.1.3.11 3.1.3.37]. The last two enzymes are known to also function in gluconeogenesis in cyanobacteria. Both of them were downregulated at least two-fold  and FBPase-II , are regulated differently in the presence of BMAA  [EC:3.1.3.11] was found only in BMAA treated samples, while fructose-1,6-bisphosphatase II/sedoheptulose-1,7-bisphosphatase  [EC:3.1.3.11 3.1.3.37] was downregulated under BMAA treatment.Four enzymes of the anabolic pentose phosphate pathway were downregulated under BMAA treatment . Among ttwo-fold . Enzyme  II, all43  and the fructose-bisphosphate aldolase, class II [EC:4.1.2.13], are under NtcA control.2  is downregulated in the way it changes in nitrogen starvation growth conditions  and aspS, aspartyl-tRNA synthetase [EC:6.1.1.12]—were found upregulated. In contrast, the phenylalanyl-tRNA synthetase β chain [EC:6.1.1.20] was slightly downregulated at BMAA presence . As it wThe BMAA treatment leads to a disturbance in the regulation of many proteins involved in nitrogen metabolism, carbon dioxide concentration mechanism, photosynthesis, amino acid metabolism, protein synthesis, and DNA transcription in cyanobacteria cells. The apparent imbalance in the cell metabolism between different metabolic processes leads to strong intracellular stress. Such internal stress was evidenced by the upregulation of four proteases, two chaperone proteins, four DNA repair enzymes, and nine stress response proteins.Nostoc PCC 7120 under BMAA treatment  was 1,5-fold downregulated at BMAA presence  in Nostoc PCC 7120 and the reason behind its downregulation caused by BMAA in diazotrophic conditions are interesting subjects for upcoming genetic and biochemical studies.Three protease proteolytic subunits of the ATP-dependent Clp protease [EC:3.4.21.92] were upshifted during the diazotrophic growth of reatment . ATP-depreatment . This prpresence . In prokNostoc PCC 7120 proteins was detected.BMAA treatment induced nine enzymes involved in stress response . These dNostoc PCC 7120 cells due to BMAA action. One of them, Glutaredoxin-3 (asl3860), which is known to have a chaperone function and take part in protein-folding catalysts, was more than fourfold upregulated under BMAA treatment  have also been found as upregulated proteins in our study  involved in high-affinity iron ion transport was downregulated in the presence of BMAA  was found upregulated  and β (nifK), as well as the downregulation of iron transport protein and an enzyme involved in sulfur metabolism under BMAA treatment ought to be the cause behind the starvation of cyanobacterial cells and the uprise of corresponding stress. Indeed, in the presence of BMAA the starvation-inducible DNA-binding protein , was found four-fold downregulated under BMAA treatment of Nostoc PCC 7120 cells in diazotrophic conditions  was found only in control samples and was not found in BMAA treated cells of Nostoc PCC 7120. It was shown that each cyanobacterial genome encodes several thioredoxins (from one to eight thioredoxins), as well as all components that are necessary for the reduction of thioredoxins [Synechocystis sp. PCC 6803 [Synechocystis sp. PCC 6803, and it was suggested that the thioredoxin-mediated redox signaling is equally significant for both oxygenic photosynthetic prokaryotes and oxygenic photosynthetic eukaryotes [Nostoc sp. PCC 73102 were predicted to be thioredoxin targets. It was found that among them were fructose-1,6-bisphosphatase, translation elongation factors, the Rubisco large subunit, chaperones, ATPase, and peroxiredoxins [https://string-db.org). The following proteins were found to be upregulated under BMAA treatment: Peroxiredoxin ; Peroxiredoxin 2 family protein/glutaredoxin  and the peroxiredoxin Q/ Bacterioferritin comigratory protein   .Synechocystis sp. PCC 6803 and its mutant that was impaired in the catalase-peroxidase activity and, therefore, was highly sensitive to oxidative stress. It was shown that hydrogen peroxide interferes with the carbon-to-nitrogen ratio status signaling in the cyanobacteria cells by reducing the intracellular concentrations of 2-OG and, consequently, changing the function of the 2-OG-sensitive global nitrogen regulator NtcA [The detoxification of reactive oxygen species (ROS) is essential for proper cyanobacteria cell functioning. It is important to note one more aspect before discussing the mechanisms of protection against oxidative stress in these photosynthetic organisms. It is known  that thetor NtcA . In thistor NtcA ,24 BMAA Summarizing the facts mentioned above, we can state that the main enzymes involved in a redox-control system of cyanobacteria cells were affected by BMAA treatment. Considering the fact that the members of the central redox machine members have cell pleiotropic functions , such asall3272) protein was found 4.5 fold upregulated in the presence of BMAA. This enzyme was upregulated three-fold in nitrogen starving cells [Nostoc PCC 7120 cells under BMAA treatment [alr0088), was found upregulated in the current study  is the LexA repressor. It represses several genes that are involved in DNA damage response (SOS response), including recA and lexA. In the presence of single-stranded DNA, RecA interacts with LexA causing an autocatalytic cleavage, which disrupts the DNA-binding part of LexA, leading to derepression of the SOS regulon and eventually to DNA repair. DNA polymerase I is also a partner of the single-stranded DNA-binding protein . This DNA polymerase exhibits both polymerase activity and 5′-3′ exonuclease activity. The Primosomal protein N’ (pri) is another protein-partner of alr0088 that is known to recognize and bind the arrested nascent DNA chain at stalled replication forks. It can open the DNA duplex via its helicase activity, promote primosome assembly and boost loading of the major replicative helicase DnaB onto DNA.The RecA . One mont study . Among int study  there aralr4504 and alr4505) .Two hypothetical proteins that may be involved in DNA metabolism and DNA recombination   were stralr4505) . For exaIt can be suggested that oxidative stress, induced by BMAA, may lead to DNA damage and, therefore, may influence the activities of both DNA cell repair and toxin-antitoxin systems.Nostoc PCC 7120 by using the National Center for Biotechnology Information (NCBI) GenBank Protein Sequence Database. The experimental discovery of hypothetical proteins in the proteome of Nostoc PCC 7120 is an essential fact, since it means that these proteins actual exist in the cells. Initially, when these proteins were annotated in the genome, their existence was only theoretically predicted. However, the proteomic method allows researchers to identify them in samples. These proteins have no resemblance to the previously characterized proteins and therefore are called hypothetical. Researchers are often disappointed when they discover hypothetical proteins in the proteome, because the functions of these proteins are unknown. We have used the existing public bioinformatics databases  in the presence of BMAA. According to the information available about its protein partners , where co-expressed genes are listed and presented  on cyanobacterium nditions . The newNostoc PCC 7120 cells was more powerful and more evident compared to the stress-responses that were previously observed under BMAA treatment in nitrogen-limited conditions [Downregulation of the nitrogen-fixation system caused by BMAA leads to N/C dis-balance that induces the decrease of both photosystem proteins. So, besides nitrogen fixation, BMAA downregulates eighteen proteins of both photosystems and light-harvesting complexes, as well as five subunits of ATP synthase and enzyme NAD(P)H-quinone oxidoreductase. As a consequence of these dramatic changes, disbalance in energy and metabolic imbalance originates in intracellular oxidative stress that leads to the activation of several key biomolecules, such as starvation-inducible DNA-binding protein, nine oxidative stress-response enzymes, four proteases, and four SOS-response and DNA repair enzymes, including RecA enzyme. This stress-response detected under diazotrophic conditions in nditions  and nitrnditions   and β (NifK) subunits of molybdenum-iron protein (component I) in nitrogenase. The corresponding genes (nifD and nifK) are under transcriptional control of the global transcriptional regulator NtcA in cyanobacteria. Furthermore, it was shown that these two genes are not the only ones under the control of the NtcA that are being affected by BMAA. BMAA impacts a total of twenty-two proteins that are under the transcriptional control of NtcA. Among them are nitrogenase structural proteins, ABC transporters, key components of photosystem II and ATPase, amino acid metabolism enzymes, carbohydrate metabolism enzymes, and peroxiredoxins. Besides that, BMAA induces a high upregulation of stress-activated proteins, such as oxidative stress enzymes  and SOS response DNA repair enzymes . This work has demonstrated for the first time that β-N-methylamino-L-alanine (BMAA) causes a severe stressful effect on cyanobacterium Thus, the obtained proteomic data shows a broad picture of regulatory changes that are induced by BMAA in the cyanobacteria metabolic networks. This information is essential for further fundamental investigations of this toxic amino acid’s regulatory roles in cyanobacteria cells. Moreover, the presented data provides new leads into the ecological impact of BMAA on microalgal communities that can be used in future investigations.Nostoc sp. PCC 7120 was grown in 100 mL Erlenmeyer flasks containing 25 mL of BG110 medium [−2s−1 and at 25 °C. Afterwards cells were collected and washed 3 times with a nitrogen-free medium (BG110). After that, cyanobacterium was grown in BG110 medium for 48 h in two experimental versions, in which: (1) the control samples were grown without the addition of aqueous BMAA solution and (2) the treated samples were grown with the addition of a water solution of β-N-methylamino-L-alanine (L-BMAA)  in a final concentration of 20 µM, as it was performed earlier in [Filamentous nitrogen-fixing cyanobacterium rlier in ,24. LateThe time period during which cells were treated with BMAA (48 h) was selected according to ,24.w/w trypsin/protein ratio and incubated at 37 °C overnight. Trypsinolysis was stopped and the acid-labile DCNa was degraded by the addition of trifluoroacetic acid (TFA)  to a final concentration of 0.5% v/v (the pH must be less than 2.0) and agitation for 45 min at 37 °C. Afterwards the sample was centrifugated at 14,000× g for 10 min to remove the DCNa. The peptide extract was desalted by using a Discovery DSC-18 Tube  according to the manufacturer’s protocol. Peptides were eluted with 1 mL 75% acetonitrile  and 0.1% TFA and dried in a benchtop vacuum concentrator SpeedVac  and resuspended in 3% acetonitrile with 0.1% TFA to a final concentration of 5 μg/μL.Lysozyme (0.3 mg/mL)  was added to each cellular pellet and gently mixed. Then the mixture was incubated for 60 min at 4 °C. Afterwards the mixture was resuspended in 100 µL of 100 mM tris-HCl buffer, pH 8.0, with the addition of Protease inhibitor Mix , 0.1% sodium deoxycholate (DCNa)  and 2.5 mM EDTA . In order to perform full cell lysis six sonication cycles of 30 s  were performed and followed by a short 5 min incubation period at 4 °C. After that, dry urea and DCNa were added to the sample to obtain the final concentrations of 8 M and 1%, respectively. After an incubation period of 20 min, the sample was centrifuged at 14,000 rpm for 10 min at 4 °C to remove intact cells. The supernatant was collected, and the protein concentration was estimated by using Bradford Protein Assay Kit . To reduce the cysteine bonds of proteins in the supernatant 5 mM Tris (2-carboxyethyl) phosphine hydrochloride (TCEP)  was added, the mixture was incubated for 60 min at 37 °C and, subsequently alkylated with 30 mM iodoacetamide  during 30 min at a room temperature in the dark. Then the step, in which TCEP was added, was repeated. Afterwards the sample was diluted 6-fold with 50 mM Tris-HCl, pH 8.0, with the 0.01% DCNa. Trypsin  was added in 1/50 v/v). Buffer B included 99.9% acetonitrile and 0.1% formic acid (v/v). Samples were loaded on a Chrom XP C18 trap column  at a flow rate of 3 μL/min for 10 min and eluted through a 3C18-CL-120 separation column  at a flow rate of 300 nL/min. The gradient was performed from 5% to 40% buffer B during 90 min followed by a 10 min period at 95% buffer B and 20 min period of re-equilibration with 5% buffer B. To wash the system and in order to prevent carryover, two blank 45-min runs, which consisted of several waves  that persisted from 5 to 8 min, were performed between the different samples.The analysis was performed on a Triple TOF 5600+ mass spectrometer with a NanoSpray III ion source  coupled with a NanoLC Ultra 2D+ nano-HPLC system  as we have described in . The HPLm/z, and the signal accumulation time was 250 ms. Ions for MS2 analysis were selected on the basis of intensity with a threshold of 200 counts per second and a charge state from 2 to 5. MS2 acquisition parameters were the following: the resolution of the quadrupole was set to UNIT (0.7 Da), the mass measurement range was 200–1800 m/z, and the signal accumulation time was 50 ms for each parent ion. Collision-activated dissociation was performed with nitrogen gas, and the collision energy ranged from 25 to 55 V within the signal accumulation time of 50 ms. Analyzed parent-ions were sent to the dynamic exclusion list for 15 s in order to get MS2 spectra at the chromatographic peak apex.The information-dependent mass-spectrometer experiment included one survey MS1 scan that was followed by 50 dependent MS2 scans. MS1 acquisition parameters were set as follows: the mass range for MS2 analysis was 300–1250 β-Galactosidase tryptic solution (20 fmol) was run with a 15-min gradient (5% to 25% buffer B) between every two samples and between sample sets to calibrate the mass spectrometer and in order to control the overall system performance, stability, and reproducibility.Nostoc sp. PCC 7120. Identification of proteins was performed with ProteinPilot  in an identification mode with the following parameters: Cys alkylation performed with iodoacetamide; trypsin digestion; TripleTOF 5600 instrument; false discovery rate (FDR) analysis; and a thorough ID search with a protein detection threshold of 95.0%. Protein identification was considered significant if the estimated local false discovery rate was 1% or lower, and at least 2 different peptides in the protein were identified with a confidence score above 95%. Spectral counting was performed with in-house script under emPAI protocol [In order to fulfill protein identification and semi-quantitative spectral counting, all LC-MS/MS data were searched against the National Center for Biotechnology Information (NCBI) GenBank Protein Sequence Database for protocol  with onlp-value with p-value thresholds of 0.05 and 0.1.Quantitative analysis was performed with MaxQuant   against https://web.expasy.org/docs/userman.html#what_is_sprot) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways database (https://www.genome.jp/kegg/pathway.html).The significantly altered proteins obtained from LC-MS/MS data analysis were subjected to analysis The proteins that were noticed to be significantly altered in the of presents BMAA according to accomplished LC-MS/MS data analysis were, therefore, subjected to analysis/analyzed/subjected to be analyzed//by using the UniProt Knowledgebase (https://stringdb.org/cgi/download.pl?sessionId=LdNVdFoNwm9Q). Gene co-expression data assembled for Nostoc (Anabaena) sp. PCC 7120 were obtained from ALCOdbCyano . The co-expression data in this database were calculated by using 116 microarray data items downloaded from the KEGG EXPRESSION Database (https://www.genome.jp/kegg/expression/). Sequence information and gene annotations were retrieved from CyanoBase (http://genome.microbedb.jp/mnt.html).Protein-Protein interactions were analyzed by the Protein-Protein Interaction Networks Functional Enrichment Analysis; http://www.collectf.org/browse/home/) [NtcA-regulated genes were found with CollecTF database (a database of transcription factor binding sites (TFBS) in the Bacteria domain) (e/home/) .


The AgNPs were characterized using ultraviolet-visible spectrophotometry (UV-Vis), dynamic light scattering (DLS), transmission electron microscopy (TEM), and Fourier-transform infrared spectroscopy (FT-IR). The anti-bacterial activity was assessed using agar well diffusion and microdilution assays, and the cytotoxicity effects on Caco-2, HT-29 and KMST-6 cells using MTT assay. The GA-synthesized AgNPs (GA-AgNPs) demonstrated higher bactericidal activity against all bacteria, and non-selective cytotoxicity towards normal and cancer cells. AgNPs reduced by NaBH4 (C-AgNPs) and the combination of GA and NaBH4 (GAC-AgNPs) had insignificant anti-bacterial activity and cytotoxicity at ≥50 µg/mL. The study showed that despite the notion that AgNPs are safe and biocompatible, their toxicity cannot be overruled and that their toxicity can be channeled by using biocompatible polymers, thereby providing a therapeutic window at concentrations that are least harmful to mammalian cells but toxic to bacteria.Silver nanoparticles (AgNPs) are the most commercialized nanomaterials and presumed to be biocompatible based on the biological effects of the bulk material. However, their physico-chemical properties differ significantly to the bulk materials and are associated with unique biological properties. The study investigated the antimicrobial and cytotoxicity effects of AgNPs synthesized using gum arabic (GA), sodium borohydride (NaBH The availability of plants makes the green method amenable to large-scale production of NPs. Over the last few years, there has been an upsurge in the application of plant-extract-reduced AgNPs on account of their immense antimicrobial efficacy, and they are perceived as future-generation therapeutic agents against drug-resistant microbes. Examples of plant-based AgNPs that have demonstrated good anti-bacterial properties and potential anticancer effects include those synthesized using Chrysanthemum indicum L [Acacia leucophloea [Ganoderma neojaponicum Imazeki [In the past, silver-based compounds were used as antimicrobial agents due to their microbicidal activities . Their bnd fungi ,3. Recennd fungi ,5, howevnd fungi . Green snd fungi ,7,8. Pland fungi ,10. The nd fungi  that canndicum L , Acacia cophloea  and Gano Imazeki  extractsAgNPs have distinct and superior properties compared to their bulk materials, and this has afforded their integration into numerous consumer  and health products to prevent microbial infestation and growth. AgNPs are now present in commercial products used daily, such as toothpaste, sunburn lotions, food packaging, medical devices, and clothing ,15. In aPterocladia capillacae, Jania rubins, Ulva faciata, and Colpmenia sinusa) [Polysaccharides have played a huge role in the application of nanomaterials, especially in biomedical applications. Polysaccharides derived from algae ( sinusa)  and plan sinusa) ) alike,  sinusa)  and GA [ sinusa) . GA is a sinusa)  and has  sinusa) . In the  sinusa) ,29, such sinusa) ,31,32, g sinusa) ,34,35, c sinusa) , quantum sinusa) , AgNPs [ sinusa) , and chi sinusa) . GA has  sinusa) ,39. GA-A sinusa)  and anti sinusa) . This stGA is a non-toxic glycoprotein polymer commonly used as a stabilizer in the food and pharmaceutical industries. It has various pharmacological properties; apart from being used as an emulsifying agent, it has antioxidant, anti-diabetic, and anti-lipid peroxidation properties, among others ,42. The Acacia senegal) had negligible flavonols, flavanols, TPC, with no antioxidant, radical scavenging or reducing abilities at 4 mg/mL, as shown in The GA species used in the current study (T and by boiling (~100 °C) solutions that contained 4 mg/mL GAE and various AgNO3 concentrations (1–5 mM). No GA-AgNPs were formed at all the tested concentrations; there was no color change in the solution at RT and a pinkish color was observed after boiling the solution (data not shown). The negligible phytochemical and lack of antioxidant contents reported for GAE in 3 to form GA-AgNPs. Other studies attempted to synthesize GA-AgNPs by devising methods to potentiate the reducing abilities of GAE, by changing the GAE pH [Synthesis of GA-AgNPs was first attempted at Re GAE pH , and by e GAE pH .3 by gum acacia [piyar gum [3 (0.1–0.5 g/40 mL) then the GAE concentrations (2–6 mg/mL/40 mL). The optimized conditions  were further used in the combined approach, with NaBH4 as an additional reducing agent, to synthesize GAC-AgNPs.A novel, greener approach, using an autoclave method, was established for other gum species to produce sterile AgNPs without additional reducing agents or change of pH. This method was successful in the reduction of AgNOm acacia , gum tram acacia  and piyaiyar gum . The sam3 was colorless before autoclaving and changed to brown after autoclaving . The optimum GAE concentration was 4 mg/mL; both 4 and 5 mg/mL of GAE gave a similar spectral profile, indicating that GA-AgNPs of the same yield, size and shape were produced by the two concentrations  to produce GAC-AgNPs (4 acting as a reducing agent to form C-AgNPs (before autoclaving) which are then capped/stabilized by GAE to form GAC-AgNPs during the autoclave process. The second assumption is that GAE and NaBH4 might have acted synergistically as reducing agents. The differences in the spectral profiles of the C-AgNPsA and GAC-AgNPs  potential, except for the GAC-AgNPs (9.33 mV). The polydispersity index (Pdi) indicated that GA-AgNPs, followed by C-AgNPs, were the most stable. Pdi serves as an indicator for the dispersity and stability of NPs, thus, NPs with a Pdi that is ≤0.05 are regarded as stable and monodispersed. Materials with a Pdi of ≥0.7 are classified as polydispersed, with broad size distribution and being less stable in suspension . The GA-3) and reducing agents (NaBH4 and GAE). The intermolecular interactions between the samples occurs via hydrogen bonding or dipole–dipole interactions during synthesis and cause shifts in the frequency or absorption of the functional groups [FT-IR was used to identify the functional groups in GAE and those that were involved in the intermolecular interactions between the precursor , 2978 , 2315, 1628 cm−1 (1650–1550 cm−1 secondary amine NH bend), and 1371 cm−1  and 1065 cm−1. The presence of different functional groups was a reflection of the phytochemical composition of the GAE; the OH bonds are attributed to alcohols or phenols and the N–H bond to amides which might be from the carbohydrates and proteins in GAE. The GAE FT-IR peaks showed similarity to those of other GA species, such as Acacia senegal and Acacia seyal [The GAE FT-IR spectra had five major absorption peaks at 3514 cmia seyal ,50,52. T+ are known for this effect and have been used for the same purposes [Burkholderia pseudomallei, the interaction and uptake of AgNPs by the bacterial species could be rapid, as the bacteria were killed within 5 min [Stability of NPs in solutions other than water is crucial for bio-applications and requires NPs that can retain their physical characteristics when introduced into a biological environment. AgNPs are usually very stable in water; however, water is hypotonic and not a suitable vehicle for bioassays . In addipurposes . Stabilipurposes . Followiin 5 min . Biologiin 5 min . The comin 5 min . Subjectin 5 min . To imprMicrobial resistance is among the leading factors responsible for death worldwide, due to the overwhelming abuse and misprescription of antibiotics ,61. OverS. aureus, MRSA, S. epidermidis, S. pyogenes) and Gram-negative  bacteria. The susceptibility of the bacteria to the treatments was assessed through agar well diffusion and broth microdilution methods. Agar well diffusion demonstrated a lack of clearing zones  in the bacteria that were exposed to MHB (negative control), GAE and GAC-AgNPs, indicating lack of anti-bacterial activity (Streptococcus mutans) [Aeromonas hydrophila and P. aeruginosa) [E. coli and S. aureus [The anti-bacterial effects of GAE and AgNPs were evaluated on Gram-positive (activity  at the c mutans)  and fishuginosa)  pathogen. aureus .E. coli which had an MIC of 25 µg/mL. The MIC values were consistent with the GA-AgNPs reported by other studies; the NPs had an MIC of 10 µg/mL in S. mutans [P. aeruginosa [A. hydrophila and P. aeruginosa, respectively [The MICs of the treatments were visually evaluated on the bacteria following microdilution assay. After 24 h treatment, GAE, C-AgNPs and GAC-AgNPs were unable to inhibit growth at all tested concentrations (6.25–100 µg/mL), as shown in . mutans , 11–45 µruginosa , 1.625 aectively . The resectively .S. pyogenes, MRSA and S. aureus  and non-cancerous (KMST-6) cells using 3--2,5-diphenyltetrazolium bromide (MTT) assay. The assay quantifies live cells by evaluating their mitochondrial metabolic activity, where live cells are able to reduce the MTT salt into the water-insoluble purple formazan. The color intensity of the dimethyl sulfoxide (DMSO)-dissolved formazan, which is measured by a spectrophotometer, is directly proportional to the amount of live cells [The cytotoxicity of the AgNPs was investigated ve cells . As show50  and breast cancer (AMJ-13) cells, while sparing the normal breast epithelial (HBL) cells [N-vinyl pyrrolidone)-coated AgNPs were not toxic to T cells at concentrations up to 50 ppm, but induced cell death of human mesenchymal stem cells (hMSCs) and monocytes at 30 and 50 ppm, respectively [The anti-bacterial effects of AgNPs have led to their use in several consumer and medical products. With an increased exposure rate to consumers who use, handle or manufacture these products, AgNPs can easily accumulate in human organs via inhalation, transdermal absorption, and ingestion . Althougo models ,16,74. Todels [+ , has beeodels [+ . Green scer AMJ-1 cells, wectively .Acacia senegal  in hot water and filtering through 0.45 µm filters. The GAE was prepared fresh before use.The GA extract (GAE) was prepared by dissolving a required amount of GA obtained from The amount of flavanols, flavonols, total polyphenolic content (TPC) and antioxidant capacity, was assessed using the ferric reducing antioxidant power (FRAP) assay kit . An oxygen radical absorbance capacity  assay and 2,2-diphenyl-1-picrylhydrazyl  assay of 4 mg/mL GAE was quantified following standard biochemical methods as previously described .The AgNPs were synthesized through: (a) chemical synthesis (C-AgNPs), (b) green synthesis (GA-AgNPs), and (c) a combined approach (GAC-AgNPs). All solutions were prepared in double-distilled water.4 (Sigma) was added to 30 mL of double-distilled water and heated to 70 °C on a heating mantle while stirring. The solution was stirred vigorously at 250 rpm. Subsequently, 20 mL of 1 mM ice-cold silver nitrate  was added dropwise into NaBH4 solution. The solution was removed from the heating mantle after a color change to yellow/brown and cooled to room temperature .C-AgNPs were prepared following a previously described method with few modification . Briefly3 in a final volume of 40 mL double-distilled water. The method was repeated, keeping the AgNO3 (0.4 g) constant and varying the concentration of GAE (2–6 mg/mL). The samples were autoclaved at 121 °C and 15 psi for 20 min and removed after 60 min when the pressure had reduced to 0 psi.GA-AgNP synthesis was adapted from a method by Venkatesham et al. . A fixed3, 4 mg/mL GAE and 10 mL of 2 mM NaBH4 in a final volume of 40 mL. The synthesis was carried in the autoclave, as described in GAC-AgNPs were synthesized following the green synthesis method  in a reaT in the dark.All the AgNPs  were washed twice and harvested by centrifugation at 9000 rpm for 30 min. The pellets were resuspended in double-distilled water and stored in amber bottles at RThe formation of AgNPs was monitored by measuring the UV-Vis spectrum of the reaction medium in the wavelength range from 300 to 650 nm using a POLARstar Omega plate reader .T. The data were represented as mean particle diameter of three measurements.The hydrodynamic size, surface charge, and Pdi of the AgNPs were analyzed by a Malvern NanoZS90 Zetasizer . The synthesized AgNPs were diluted 5-fold with double-distilled water; 1 mL aliquots were sampled in DLS cuvettes or DS1070 zeta cells  and examined for size distribution and zeta potential, respectively. The particle diameters were assessed at a scattering angle of 90 °C at R−1. The baseline corrections were performed for all spectra.The infrared spectra of absorption or emission of the AgNPs and GAE in solution were identified using a Perkin Elmer Spectrum Two FT-IR spectrophotometer  in the wavelength range 4000–500 cmHRTEM analysis was performed by the addition of a drop of each AgNP solution on carbon-coated copper grids, then left to dry under ambient conditions. The shape and size of AgNPs were analyzed using TecnaiF20 HRTEM  with an accelerating voltage of 300 kV at the Electron Microscope Unit . In addition, the core size distribution of the AgNPs was calculated using ImageJ software .The stability of AgNPs over time was evaluated following a previous method , by measKlebsiella pneumoniae (K. pneumoniae), Escherichia coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa), Staphylococcus aureus (S. aureus), Methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus epidermidis (S. epidermidis), and Streptococcus pyogenes (S. pyogenes). All the bacterial strains were purchased from American Type Culture Collection . The anti-bacterial activity was determined by agar well diffusion and microdilution assays according to the standards set by the Clinical and Laboratory Standard Institute with few modifications [The anti-bacterial activity of the AgNPs was evaluated using Gram-negative and Gram-positive bacterial strains; i.e., ications .6 CFU/mL by diluting the 0.5 McFarland turbid suspensions to 1:150.Bacterial colonies were cultured in MHB while shaking at 120 rpm overnight at 37 °C, then diluted at 1:100 in fresh MHB and cultured until reaching a 0.5 McFarland turbidity standard prior to experiments. The bacteria were used for anti-bacterial tests at 1.5 × 106 CFU/mL using sterile cotton swabs. Wells of 6 mm diameter were made on the MHA plates, to which 20 μL of the AgNPs were added. The MHA plates were then incubated overnight at 37 °C. Ciprofloxacin  was used as a positive control. The anti-bacterial activity of the AgNPs was determined by the presence of clear zones surrounding the wells. The diameter of the clear zones was measured using calipers after 24 hr.The bacterial cultures were streaked on Mueller–Hinton Agar  plates at 1.5 × 10Microdilution assay was used to determine the minimum inhibitory concentration (MIC) ,49 and m6 CFU/mL) were exposed to different concentrations (0–100 μg/mL) of the AgNPs and incubated at 37 °C for 24 hr. The MIC values were visually observed, followed by measuring the optical density (OD) of the bacterial culture at 600 nm using a POLARstar plate reader. Bacterial growth was further evaluated by Alamar Blue colorimetric assay , where 10 μL of the dye was added to each well and incubated for 3 hr. The blue color was converted to a pink-purplish color by live bacteria that was quantifiable by measuring absorbance at 570 nm and a reference wavelength at 700 nm [The MIC of the AgNPs required to inhibit the visual growth of the bacteria was determined according to the microdilution method ,49. The t 700 nm .The MBC of the AgNPs was determined in the bacteria used for MIC, where a loopful of broth from the wells was spotted onto fresh MHA and incubated at 37 °C for 24 hr. The lowest concentration that exhibited no growth on the MHA was considered as the MBC.5 cell/mL density, 100 µL in each well and incubated for 24 hr. The cells were treated with 0–500 μg/mL of the AgNPs and extracted in triplicates. The cell viability was assessed by adding 10 μL of 5 mg/mL of MTT (Sigma) solution to each well and incubated for 3 hr. Later, the MTT solution was discarded and 100 μL of DMSO was added to each well. The absorbance of the formazan product was measured at 570 nm with a reference at 700 nm using a POLARstar Omega plate reader. The concentration that inhibited 50% cell growth (IC50) was further analyzed by Graphpad Prism 6.0.The effect of the AgNPs was evaluated by MTT assay, as previous described, on the human cell lines, KMST-6 normal skin fibroblasts, HT-29 and CaCo-2 colon carcinoma cells . The celp-value of <0.05 was considered statistically significant.All the experiments were carried out in triplicate and the results were analysed using Graphpad Prism 6.0. The data are presented as means ± SD according to one-way ANOVA test followed by a post hoc, multiple comparisons (Tukey’s) test. A The growing interest in medical application of AgNPs has warranted greener methods for their synthesis to prevent toxicity and improve biocompatibility. Synthesis of AgNPs using plant extracts presents not only a greener method but a sustainable, reproducible and upscalable approach. However, for biomedical applications, the safety of plant-synthesized AgNPs must be authenticated. The current study demonstrated that GAE alone, and in the presence of a chemical reducing agent, produced AgNPs with distinct bioactivities. The GA-AgNPs demonstrated broad spectrum anti-bacterial effects on both Gram-positive and Gram-negative bacteria, and non-selective cytotoxicity on normal and colon cancer cells in the same concentration range. Interestingly, these effects were reduced in the GAC-AgNPs, suggesting that surface coating can be used to channel the effects of AgNPs. The selective and reduced cytotoxicity demonstrated by GAC-AgNPs towards colon cancer cells demonstrated that surface composition can be used to control the biodistribution, uptake and efficacy of AgNPs. AgNPs represent the next generation of antimicrobial agents, and have potential to help solve the antimicrobial resistance problem. Their biocompatibility can be enhanced by modifying the surface of AgNPs with targeting molecules or biocompatible molecules, such as PEG, for medical application.


The highly contagious nature of SARS-CoV-2 has led to several studies on the transmission of the virus. A little studied potential fomite of great concern in the community is currency, which has been shown to harbor microbial pathogens in several studies. Since the onset of the COVID-19 pandemic, many businesses in the United States have limited the use of banknotes in favor of credit cards. However, SARS-CoV-2 has shown greater stability on plastic in several studies. Herein, the stability of SARS-CoV-2 at room temperature on banknotes, money cards and coins was investigated. In vitro studies with live virus suggested SARS-CoV-2 was highly unstable on banknotes, showing an initial rapid reduction in viable virus and no viral detection by 24 hours. In contrast, SARS-CoV-2 displayed increased stability on money cards with live virus detected after 48 hours. Environmental swabbing of currency and money cards on and near the campus of Brigham Young University supported these results, with no detection of SARS-CoV-2 RNA on banknotes, and a low level on money cards. However, no viable virus was detected on either. These preliminary results suggest that the use of money cards over banknotes in order to slow the spread of this virus may be ill-advised. These findings should be investigated further through larger environmental studies involving more locations. They primarily infect a wide range of animals and are most ecologically diverse among bats, with viruses from the Alphacoronavirus and Betacoronavirus genera known to infect humans. The SARS-CoV-2 virus likely originated from the SARS-like virus in the Rhinolophus bat family [Sarbecovirus subgenus [Betacoronaviruses, are notably severe exceptions to this rule [Coronaviruses are large, enveloped, positive-strand RNA viruses that belong to the t family  and fallsubgenus ,6. Thoughis rule –9. Althohis rule ,11.SARS-CoV-2 transmission patterns. Although primarily transmissible from person-to-person through respiratory droplets, evidence shows the virus is also transmissible through direct contact, feces, or other body fluids [SARS-CoV-2 has been shown to be transmissible by asymptomatic carriers even during its incubation period [SARS-CoV-2 on various surfaces [3.7 per mL media down to 100.6 per mL media) [To curb future outbreaks, both public health and molecular perspectives are essential to understand y fluids –15. SARSn period ,17. Thisn period . Fomitesn period . Van Dorsurfaces . Five enL media) . In contL media) .SARS-CoV-2 RNA, including aerosols [In environmental, real world studies, a variety of substances and surfaces have been tested for the presence of aerosols ,20–27 anaerosols ,25,27–29aerosols  as well aerosols  in Chinaaerosols , with poaerosols ,24,26 (raerosols ). Lack oS. aureus on banknotes recovered from hospitals, and S. aureus, Salmonella and E. coli detected on banknotes recovered from food outlets [SARS-CoV-2 was modeled in the laboratory on four common surfaces including swine skin, uncirculated United States of America $1 and $20 Federal Reserve notes, and scrub cloth, which served as a common clothing example [SARS-CoV-2 on credit card or coin has not been reported, and there has been a lack of environmental testing outside of the laboratory, with the exception of a study in Bangledesh reporting detection of SARS-CoV-2 RNA on approximately 7% of circulating banknotes [SARS-CoV-2 in the U.S.A., the stability of the virus on U.S.A $1 banknotes, coin and money cards in the laboratory is studied herein. In addition, the frequency of detection of SARS-CoV-2 on environmental paper money and money cards was assessed in the Brigham Young University (BYU), Provo, Utah area.A little studied potential fomite of great concern in the community is currency ,35, whic outlets . Lower d outlets . In a re example . At roomanknotes ,39. In oFor environmental samples, all environmental positive samples collected in 50% DMEM were disposed of in Biohazard bins after the addition of an equal volume of 70% ethanol. All assayed money cards were immediately disinfected after use with 70% ethanol. All work involving live SARS-CoV-2 was performed in the biosafety level-3 facility at BYU, Provo, UT. All work was conducted under proper safety handling procedures approved by the BYU Institutional Biosafety Committee (IBC-2020-0068). Verbal consent was obtained from participants  and consent was waived by the ethics committee (no names or places were recorded with sample information).2. For 24-well plate preparation, cells were seeded at 200,000 cells per well in DMEM with 10% FBS and used for assays 18–24 hours later.Viral cultivation and plaque assays were performed using African green monkey kidney  cells obtained from American Type Culture Collection (ATCC). VERO cells were maintained in T-75 flasks containing Dulbecco’s Modified Eagle’s Medium  supplemented with 10% Fetal Bovine Serum  at 37°C, and 5% CO−1 through 10−10 by 10-fold serial dilutions in DMEM. VERO cells in 24-well plates were then inoculated with 200ul of a virus dilution. Plates were placed at 37°C and 5% CO2 for 1 hour and manually rocked every 10 minutes. A 1ml-overlay of 1:1 mixture of 2X Minimal Essential Medium  with 8% FBS and 1.5% low melting agarose  was placed in the wells. Agarose plugs were allowed to solidify at room temperature for 30 minutes. Plates were then incubated for 72 hours at 37°C and 5% CO2. Post incubation, wells were fixed with 10% formaldehyde for 1 hour. Agar plugs and formaldehyde were then removed and wells were washed twice with distilled water. 200ul of crystal violet stain (0.4%) were then placed in each well for 3 minutes at room temperature, followed by rinsing with distilled water twice and air drying. Plaques were counted and titers were performed in duplicate.SARS-CoV-2 (2019-nCoV/USA-WA1/2020) was obtained from the ATCC. Viral titer was obtained by plaque assay in VERO cells. Briefly, stock virus was diluted 1:2 and from 105−107 cells directly in the culture dish and pipetting up and down several times to homogenize. Samples were allowed to incubate for 5 minutes, and then centrifuged for 5 minutes at 12,000 ×g at 4–10°C. The clear supernatant was transferred to a new tube and stored at -80°C.SARS-CoV-2 infected VERO cells were lysed by adding 0.3–0.4 mL of TRIzol Reagent  per 1 X 10Due to the reduction in usage of cash in and around campus, environmental samples of cash were primarily obtained from the Brigham Young University vault, a daily collection of currency and coin from university-based stores, restaurants, dormitories and vending machines. In addition, fresh samples were obtained from local restaurants and assayed within one hour. The entire surface of cash and/or coin was swabbed around the edges of the bill and then back and forth across the surface for ~10 seconds with a sterile cotton swab moistened with sterile 50% DMEM . The swab was immersed with agitation in 500uL 50% DMEM. The samples were immediately assayed for viral signatures within one hour after collection using a loop-mediated isothermal amplification (LAMP) assay. The WarmStart LAMP Kit  using SARS-CoV-2 specific primers  or the S7 pfu/mL) was inoculated onto circulated (used) U.S currency; $1 U.S.A. banknotes, credit card, quarter, and penny (three of each) and incubated in an operating biosafety cabinet at 22°C. Currency was sampled at the following four post-inoculation time points: 30 minutes (used as time zero) then 4, 24, and 48 hours. Negative controls were inoculated with 20ul of sterile DMEM. At each time, cotton swabs were dipped in 500ul DMEM contained in 1.5 ml microcentrifuge tubes and used to swab area where virus was inoculated. Swabs were immediately placed back into the collection microcentrifuge tube of 500ul media and mixed thoroughly to extract virus. Samples were then serially diluted; 50ul sample into 450ul DMEM from 10−1 to 10−4 and assayed for infection of VERO cells as described above along with a DMEM only negative control. All tests were performed in duplicate, however not all samples were able to be counted due to the health of individual plates. Previously circulated $1 U.S.A. banknotes, credit cards and coins were sterilized by U.V. light prior to use in this study.Viable SARS-CoV-2 virus . Half-life was estimated from the plot of the log2 of the titer, using JMP Pro to fit a line and deriving the half-life from the slope according to the equation (-log2 2/slope). Time points after a time point approximating a zero titer were dropped from the linear fit.Graphs were produced using GraphPad Prism software version 8.0.0  and ANOVA with post hoc Tukey HSD was performed using JMP pro version 15 . Further significant reductions in viable virus occurred at the 24 and 48 hour time points, where no live virus was detected . In contrast, money cards displayed only a 10-fold or 90% reduction in titer at 30 minutes  which may in part be due to a difference in the ability of the viral suspension to soak into or bind plastic money cards versus paper banknotes. Further significant reductions in titer occurred at 4 hours , however live virus was still detectable at 24 and 48 hours post inoculation, displaying a 99.96% and 99.97% reduction, respectively, when compared with time 30 minutes . Quarters and pennies were similar to the money card, with a stronger initial reduction in viral titer  and virus detectable at the 24 and 48 hour time points. Thus, viable SARS-CoV-2 appeared to be most stable on plastic money cards, with banknotes providing the least stability of all four surfaces tested in this study.Viable SARS-CoV-2 was spotted on $1 banknotes, quarters, pennies, and credit cards and then extracted by swabbing with a MEM-moistened sterile swab at time points 30 minutes  as well as 4, 24, and 48 hours post inoculation. Extracted samples were immediately assayed for viable SARS-CoV-2 via plaque counts in VERO cells . SARS-CoAll samples displayed an initial rapid drop in viable virus (PFU/mL) followed by a different rate of decay. This may be due to surface drying times. Therefore, modeling of the half-life of SARS-CoV-2 on these surfaces was conducted with and without inclusion of the starting titer to obtain two-step decay rates, initial rates (from time 0 to time 0.5 hours) of decay versus later rates  ID money cards, were swabbed and assayed via SARS-CoV-2 LAMP assay , Table 2Four hundred and twenty-nine U.S.A. banknotes from the BYU campus vault (or nearby restaurants) were also tested for the presence of SARS-CoV-2 RNA. No sampled banknotes tested positive, consistent with the observed instability observed on banknotes . Only onIn the United States, paper currency is a blend of ~75% cotton and 25% linen with ample surface area and it is routinely passed between individuals suggesting that it may function as a fomite in the spread of many bacterial and viral diseases. When the coronavirus pandemic was announced, a common reaction from businesses was to stop all use of U.S.A. banknotes to hypothetically reduce the spread of this virus. Neither the World Health Organization (WHO) nor the Centers for Disease Control (CDC) in the United States has banned the use of paper money and coin, but they have emphasized that people should wash their hands after handling money as a precaution. Many stores are still either limiting or completely denying the use of cash. Our results suggest that SARS-CoV-2 is more stable on plastic money cards, with only a 99.6% reduction of the virus after four hours, compared with a 99.9993% reduction of the virus on $1 U.S.A. banknotes immediately after inoculation of U.V.-sterilized, circulated banknotes . These rOur results are consistent with reports that SARS-CoV-2 displays increased stability on plastic when compared with cotton and support the recommendation that paper sheets and bags be used instead of plastic to reduce the spread of COVID-19 ,37,42–45In contrast to cotton bank notes, money cards did not show this drastic initial decrease in our study, but instead showed a gradual decrease to 24 hours, followed by low level stability which may be due to increased time necessary for drying on the surface. The hypothesis that the decrease in stability on paper or cotton bank notes versus plastic is due to dryness of the paper and porous solids is unproven, and appears to contradict the experimental studies showing higher reduction in coronaviruses numbers at high humidity (80% versus 20%) . Thus, iThe conclusions herein suggest one clear shortcoming of the study herein and several others—that the use of cotton swabs to retrieve virus from samples may inactive the virus, allowing only comparison of different surfaces in these studies. SARS-CoV-2 clearly has differential stability on different money surfaces which may explain reported differences in banknote such as the Austrailian bank notes versus the U.S.A. and Euro. Clearly further study is necessary to understand the binding and stability properties of SARS-CoV-2. In any case, transmission of SARS-CoV-2 by U.S.A. banknotes appears unlikely, requiring a high initial viral load and rapid transfer to another individual in order to avoid viral binding and/or inactivation.S1 Raw images(PDF)Click here for additional data file.


BackgroundThe standard four-port laparoscopic cholecystectomy (LC) is the gold standard procedure. The various clinical trials and reports in the literature have suggested that the three-port technique LC can be done safely with acceptable results.Still, that the three-port LC offers any added benefits to the patient is a controversial issue especially in view of safety and feasibility. In this study, we report the experience of three-port LC compared to four-port LC technique, its safety, feasibility and outcomes.Materials and methodsA prospective randomized study was conducted between two groups which included 165 cases - 93 patients were included in three-port LC (Group A) and 72 patients in four-port LC (Group B). Operative time, intraoperative complications, postoperative pain, length of hospital stay, analgesics requirement, conversion to open and return to normal activities were parameters of evaluation.ResultsDemographic data was comparable in both the groups. Three-port LC Group A had lesser post-operative pain and analgesics requirements. The mean postoperative pain visual analogue scale (VAS) score on day 1 was (4.16 and 6.24), on day 7 was (1.26 and 1.81) in three-port group and in four-port LC group, respectively. The mean days of analgesics requirement were 2.56 days and 4.21 days among three-port group and four-port group, respectivelyLength of hospital stay was less and returning to work was early in three-port group. There was no statistical difference in operative time. The mean operative time among three-port LC group A and four-port LC group B was 36+/-8.6 minutes (30-68) and 39+/-7 minutes (30-90), respectively. The overall outcomes were comparable to four-port LC.ConclusionThree-port LC is a feasible and safe procedure for LC with satisfactory outcomes like lesser postoperative pain, postoperative stay and less scars, when performed by experienced hands, especially in acute cholecystitis. The use of fourth port should be done when required in a difficult situation. Standard four-port laparoscopic cholecystectomy (LC) is considered to be a gold standard technique for cholecystectomy , 2.Various modifications have been done in the four-port laparoscopic cholecystectomy like decreasing the number and size of the ports to reduce the postoperative pain and better cosmetic results -6. The uThese studies have mentioned that three-port LC outcomes need to be re-evaluated by other large sample studies.These modifications have shown reduced postoperative pain and less use of analgesics. Some surgeons have reported the use of two ports and mini-instruments for doing LC ,10,11.The present study was conducted to compare the feasibility and safety of three-port laparoscopic cholecystectomy as compared to four-port LC. The outcome was measured based on operative time, intraoperative complications, conversion to four-port or open procedure, postoperative pain by visual analogue scale (VAS), length of hospital stay, resuming normal activities and overall satisfaction of the patient.A randomized prospective study was carried out at a tertiary care teaching hospital in central India, between August 2017 and August 2020. A total of 165 patients who required laparoscopic cholecystectomy between the age group 18-75 years were included in the study after the Narendra Kumar Prasadrao Salve ethical committee approval. Informed consent was taken from all the patients included in the study. Patients were divided into two groups - three-port LC (Group A) had 93 patients and four-port LC (Group B) had 72 patients. Operative time, complications, postoperative pain, length of hospital stay, analgesics requirement, conversion to open and return to normal activities were evaluated in both the groups.The data regarding demographic characteristics, clinical history, examination findings, and investigations, intraoperative and post-operative findings was collected using pre-validated, semi-structured, standard case record proforma.The three-port technique was performed by creating pneumoperitoneum using Veress needle technique through a supra-umbilical stab incision through which a 10-mm camera port  was introduced. An operating telescope  was introduced from the supra-umbilical camera port. Another 5-mm trocar  was inserted 3 cm below the xiphisternum, and after evaluation of the gall bladder position and anatomy, a 5-mm working port  was introduced at the right hypochondrium anterior axillary line 3 cm below the subcostal margin. The operating surgeon and assistant stood on the left side of the patient while the staff nurse stood on the right side of the patient. The monitor, insufflation and light source system were kept on the right side of the patient. The gall bladder infundibulum was held and retracted towards the right upwards direction from the right subcostal 10-mm port. A 5-mm epigastric port was used for Calot’s triangle dissection using blunt and sharp hook intermittently. After doing the posterior dissection, a critical view of safety was seen. Cystic artery and cystic duct were dissected in Calot’s triangle. The cystic duct was then clipped with a 5-mm clip applicator and divided followed by the cystic artery. Intraoperative cholangiogram (IOC) if indicated was performed through the 5-mm epigastric port by using a fluoroscope system. After the dissection of the gall bladder from the gall bladder fossa in proper plane, the gall bladder was separated. The specimen was retrieved from the 10-mm supra-umbilical port using the 5-mm telescope from the epigastric port under vision. After achieving the proper hemostasis, trocars were removed under vision. Port sites were sutured with Vicryl 3-0 cutting needle suture Figures .The four-port LC was performed using the fourth port at the right subcostal region introduced as per the convenience and position of the gall bladder for retraction of fundus of the gall bladder. The rest of the procedure was the same as three ports LC.After surgery patients were shifted to the postoperative care unit. After complete stabilization, the patients were transferred to the ward where analgesics were administered (paracetamol and/or diclofenac) unless allergies or specific contraindications were noted. Patients received their analgesics according to their pain ratings measured by nursing staff using the visual analogue scale. The total amount of analgesia required by each patient was calculated over 48 hours post-surgery or till the patient was discharged. The patient was discharged after the consensus in the surgical team, nursing staff and the patient regarding the recovery. Ports dressing was checked for any bleeding or hematoma. Post-discharge, the patients were prescribed antibiotics and analgesics with advice for follow-up in OPD after one week.Statistical analysisContinuous data is presented in form of mean ± SD and compared using independent t-test whereas categorical data is presented in frequency (%) and compared using Chi-square test. Statistical software named “MedCal - 12.2.1” was used for analysis. Significance is set at 5% in this study.In the present study demographic characteristics among Group A three-port LC (n=93) and Group B four-port LC (n=72) were compared. The mean age of the study subjects was 42.52 +/- 3.7 years in Group A and 46.37 +/- 15.01 years in Group B . On gender-wise distribution of the study subjects, it was observed that the majority were females in both study groups, however, in Group A ratio of females was greater .In the current study preoperative diagnosis was assessed. It was observed that the majority had symptomatic cholelithiasis (81.72% and 86.11% in either group), followed by acute cholecystitis  and gall bladder polyps . In this study, previous endoscopic retrograde cholangiopancreatography (ERCP) due to choledocholithiasis was done among 5.38% and 9.72% patients in both Group A and Group B, respectively. The difference was not statistically significant .On the assessment of previous upper abdominal surgery among the study subjects, no statistically significant difference was observed among both the groups . Table In the present study, intraoperative and postoperative variables were studied as shown in Table Signs of acute cholecystitis were not significantly different among both the groups. Gall bladder perforation during dissection and stone spillage were reported more in three-port LC group as compared to four-port LC group. There was no statistically significant difference found in Calot’s triangle adhesions for both the groups.Anatomical variations were noted in five patients (5.37%) and six patients (8.33%) in three-port LC Group A and four-port LC Group B, respectively . Conversion to open surgery was observed among one patient (1.08%) and two patients (2.78%) in Group A and B, respectively . Drain was placed among seven patients (7.53%) and five patients (3.6%) in Group A and B, respectively . Port site bleeding was reported among two patients (2.15%) and three patients (4.17%) in Group A and B, respectively . Conversion to four ports from three-port LC was observed among four patients (4.3%).The mean postoperative pain VAS score on day 1 was 4.16 and 6.24, day 7 1.26 and 1.81 in three-port group and in four-port LC group, respectively . Mean days of analgesics requirement were 2.56 days and 4.21 days among three-port group and four-port group, respectively . Mean hospital stay duration was 3.12 days and 4.86 days among three-port group and four-port group, respectively . Mean days of return to normal activities were 4.5 days and 6.2 days among three-port group and four-port group, respectively. Mean satisfactory score after seven days was 7.6 and 6.4 among three-port group and four-port group, respectively . Port sites infection was observed among one patient (1.07%) and two patients (2.14%) among three-port group and four-port group, respectively. Postoperative jaundice was observed in one patient (1.08%) in three-port group.In the current study, we assessed the reasons for conversions of patients from three-port LC to four-port LC or to open procedure as shown in Table Four-port laparoscopic cholecystectomy is the gold standard procedure for LC. The aim of the laparoscopy procedure includes decreased pain, improved cosmetic results and decreased duration of hospital stay compared to laparotomy. Over a period of time, LC has been modified and developed in many ways including reduction in size and number of ports for the benefit of the patients as reported in the literature -6,12,13.Four-port LC is most commonly used, as it has better anatomic views and is easier to learn. The use of the fourth port in LC has been questioned by many experienced surgeons and there are several literature reports which mention that the use of fourth port can be avoided and LC can be done with three ports without compromising patient’s safety , 8. CercA meta-analysis study by Sun et al. , which cThe technique of LC is modified over a period of time to achieve better results. The fourth port has been used in LC to retract the gall bladder fundus for better visualization of biliary anatomy, Calot’s triangle and to achieve the critical view of safety during LC .In this study comparison of outcome measures, feasibility and safety of three-port LC as compared to four-port LC in acute as well as chronic cholecystitis cases has been done. There were no bile duct injuries or any major complications in either of the patients. Port site bleeding/hematomas were seen less frequently in patients who underwent three-port LC, however, the difference was not statistically significant. The conversion rate, when compared with four-port LC, did not change as shown in the literature as well , 17.In the present study, we also compared demographic characteristics among the two groups. The mean age of the study subjects was 42.52 ± 3.7 years in Group A and 46.37 ± 15.01 years in Group B . In the gender-wise distribution, the majority of subjects were females in both study groups, however, in Group A proportion of females was greater . In the current study, we assessed preoperative diagnosis, where symptomatic cholelithiasis (81.72% and 86.11% in either group), acute cholecystitis  and gall bladder polyps  were reported. Previous ERCP due to choledocholithiasis was done among 5.38% and 9.72% of study subjects in respective groups, and this observation was not statistically significant . When previous upper abdominal surgery was assessed among the study subjects, significantly different observation among either study group was noted (3.23% and 8.33% in either group) .In our study, the mean operative time among three-port LC Group A was 36 ± 8.6 (30-68 min) and in Group B four-port LC it was 39 ± 7.4 (30-90 min). The operative time was relatively lesser but not significantly different  in three-port group. Similar durations of operative time were reported in various studies in the literature in comparison between three-port LC and four-port LC .The operative time in three-port LC is comparatively shorter in comparison with four-port LC due to more time consumed in insertion and closure of fourth port in four-port LC which sometimes may need hemostasis. Moreover, in four-port LC the operator spends more time in adjusting the fourth port instrument used to retract the gall bladder fundus which is usually handled by the 2nd assistant or the nursing staff which is not there in three-port LC, where it's done by the surgeon himself.Signs of acute cholecystitis were not significantly more among three-port LC group as compared to four-port LC group. Gall bladder perforation during dissection, bleeding due to slippage of clip and stone spillage were reported more among three-port LC group as compared to four-port LC group but not statistically significant. Calot’s triangle adhesions were not found to be significantly different in either group.Anatomical variations were observed in five (5.37%) and six (8.33%) study subjects in Group A and B, respectively . Conversion to open surgery was observed among one (1.08%) and two (2.78%) study subjects in Group A and B, respectively . The drain was placed among seven (7.53%) and five (3.6%) study subjects in Group A and B, respectively . Port site bleeding was reported among two (2.15%) and three (4.17%) study subjects in Group A and B, respectively . Conversion to four-port from three-port LC was observed among four (4.3%) patients.The mean postoperative pain VAS score on day 1 was 4.16 and 6.24, day 7 1.26 and 1.81 in three-port group and in four-port LC group, respectively. Mean days of analgesics requirement was 2.56 days and 4.21 days among three-port group and four-port group, respectively. Mean hospital stay duration was 3.12 days and 4.86 days among three-port group and four-port group, respectively, Mean days of return to normal activities was 4.5 days and 6.2 days among three-port group and four-port group, respectively. The mean satisfactory score at seven days was 7.6 and 6.4 among three-port group and four-port group, respectively.As reported in other studies, the present study also showed comparable less postoperative pain, less analgesics requirements, less number of days of hospitalization and early resumption of work in three-port LC as compared to four-port LC , 7-8, 18However, there was no statistical difference in the postoperative pain and complications on day 7 in both the groups.Port sites infection was observed among one (1.07%) and two (2.14%) patients among three-port group and four-port group, respectively. There was post-operative jaundice in one patient (1.08%) in three-port group which was managed by ERCP and bile duct stenting.In the present study, we have assessed the conditions which required conversions from three-port LC to four-port LC or to open cholecystectomy which is rarely reported in the previous literature. Calot’s triangle adhesions due to severe acute cholecystitis and bleeding were the main reasons for the conversions. Four patients (4.3%) required conversion from three-port LC to four-port LC. One patient (1.08%) from three-port LC and two patients (2.78%) from four-port LC were converted to open cholecystectomy due to difficult Calot’s triangle dissection and bleeding, keeping the low threshold for conversion. Other reasons for conversions were bile spillage, abnormal anatomy and displacement of clip in one (1.07%) patient.All the procedures were performed by two experienced surgeons. The conversion rates were on the higher side but not significantly different from the other studies, maybe because the low threshold for conversion was kept. There was no incidence of bile duct injury or any major complications , 18-19.After the introduction of LC, there were concerns regarding more rate of biliary injuries . VariousToday in the era of minimally invasive surgery the main advantages of LC are significant less postoperative pain, less hospital stay, early resumption of routine activities and better cosmoses. Various studies in the literature have shown that all these can be achieved better by reducing the size and the number of the ports without compromising the patient’s safety , 21-24.This study has the limitation that the sample size was not large enough. We had a larger rate of conversion to four-port as the threshold for conversion to convert to four ports or to open procedure was kept at low in the interest of patients' safety.In this study, we conclude that the use of three ports in LC is feasible and can be performed safely with acceptable and comparable outcome measures as in routine four-port LC. We can achieve better results with added advantages of minimally invasive surgery offered to the patients by using the three-port technique of LC without compromising the patient’s safety. It is advised to do a similar kind of prospective study with a larger sample size. However, it should be recommended that three-port LC be performed by experienced hands and keeping a low threshold for the fourth port insertion or open conversion whenever indicated in a difficult situation.


A phase III randomized multicenter trial (ALTER0303) reported anlotinib to be significantly beneficial to patient survival. An array of inflammatory biomarkers, such as neutrophil lymphocyte ratio (NLR) and platelet lymphocyte ratio (PLR), are associated with the response to treatment in numerous types of cancer. However, we found few studies investigating the predictive value of NLR or PLR in advanced NSCLC treatment with anlotinib. Thus, our objective was to examine the relationship between NLR and PLR and treatment effect, as well as to individuate patient stratification and selection.  NLR and PLR as well as their variations were calculated in 152 advanced NSCLC patients receiving anlotinib as a third or further-line treatment at Ningbo Medical Center Lihuili Hospital between July 2018 and December 2020. The best cut-off values of NLR and PLR for predicting the treatment response were selected. Survival curves were plotted using the Kaplan–Meier method, while univariable and multivariable Cox regression were used to identify and determine dependent and independent predictors of survival. P = 0.007), high pre-PLR (P = 0.004), and elevated post-NLR (P = 0.010). Multivariate analysis determined high pre-PLR (>205.63) and elevated post-NLR to be independently associated with poor PFS or OS. Patients whose risk score was 2 resulting from the prediction model based on pre-PLR and post-NLR had a 4.52 times higher risk of death compared to patients whose risk score was 0 . , Low disease control rate (DCR) was related with a high pre-NLR ( Pre-PLR and post-NLR were independent prognostic indicators in patients with advanced NSCLC receiving anlotinib as a third or further-line treatment. Patients whose risk value score was 0 had a higher therapy effectiveness and better survival. Lung cancer is one of the most common malignancies and a leading cause of cancer-related death worldwide.Non-small cell lung cancer (NSCLC) accounts for 85% of all lung cancers, a majority of which present with advanced metastatic disease , 2. The Previous studies have demonstrated that the systemic infection response plays a critical role in the symptom response , 9. ManyIn this study, we retrospectively analyzed the prognostic and predictive role of NLR, PLR, and their variations in advanced NSCLC patients receiving anlotinib as a third or further-line treatment. The aim is to stratify and select individual, potentially reliable and convenient markers for patients.We retrospectively reviewed data of patients with advanced NSCLC receiving anlotinib as a third or further-line treatment at Ningbo Medical Center Lihuili Hospital between July 2018 and December 2020. The inclusion criteria were as follows: (1) a pathology diagnosis of stage IV NSCLC (recurrent or metastatic); (2) receiving at least two standard systemic therapeutic regimens; (3) treatment with anlotinib as a monotherapy for more than 2 weeks; (4) no obvious symptoms of infection or recent treatment with steroids; (5) no history of liver cirrhosis; and (6) available NLR and PLR data starting from 2 weeks before treatment to 2-4 weeks after treatment. A total of 152 patients were enrolled.Clinical characteristics and pathological findings, such as age, performance status, pathology, and history of tumor surgery, were recorded. Hematology data collected from 2 weeks prior to treatment to 2-4 weeks after treatment including neutrophil count (×109/L), platelet count (×109/L), and lymphocyte count (×109/L). We define the NLR as a neutrophil count divided by lymphocyte count, and PLR as a platelet count divided by lymphocyte count. Pretreatment NLR (pre-NLR) was determined within 2 weeks before treatment, while posttreatment NLR (post-NLR) within 2-4 weeks after treatment. Similarly, pretreatment PLR (pre-PLR) was recorded within 2 weeks before treatment and posttreatment PLR (post-PLR) within 2-4 weeks post treatment.Anlotinib was indicated for advanced NSCLC patients as a third or further-line treatment with an initial oral dose of 10-12 mg/day. Each cycle was defined as 2 weeks on-treatment followed by 1 week off-treatment. In case of an intolerable treatment-related adverse event, the dose would be cut back to 8-10 mg per day. Treatment was continued until disease progression or treatment intolerance.Follow-up evaluations, including B-ultrasound and computed tomography scan, were performed at 3 or 6 weeks after introducing anlotinib based on the Response Evaluation Criteria in Solid Tumors (RECIST) criteria. Systemic review was performed per every 2 cycles. Diagnostic examinations were performed when recurrence was suspected. DCR was defined as the percentage of patients evaluated who achieved complete response (CR), partial response (PR), and stable disease (SD). ORR was defined as the percentage of patients evaluated who achieved CR and PR. Progression free survival (PFS) was defined as the time from the onset of therapy and to progression or last time of follow up. The overall survival (OS) was calculated from the start of treatment to the time of death (irrespective of cause of death) or to last follow-up date.The statistical analysis was performed with SPSS  and R version 4.0.1 . The best cut-off values of NLR and PLR from the maximum Youden's index, as well as the corresponding sensitivity and specificity for discriminating DCR and progressive disease (PD), were obtained from receiver operating characteristic (ROC) curve analysis. Using Fisher's exact or chi-square test, Kaplan–Meier method to plot survival curves and determine any differences by the univariable and multivariable Cox regressions. Variables with P value of <0.05 in the univariable analysis were considered for inclusion in the multivariable logistic regression model. The model was validated by the 5-fold cross-validation. Threshold for statistical significance was set at P < 0.05.A total of 152 patients who met the inclusion criteria were chosen to be included in our study. The basic patient characteristics are summarized in With a median follow-up time of 7.05 months (1.1 to 24.4 months), 121 patients (79.6%) had died and all the patients were recrudescent at the end of the follow-up time. 10 (6.6%), 98 (64.5%), and 44 (28.9%) patients achieved PR, SD, and PD, respectively. None of them achieved a CR. DCR was achieved in 71.1% and ORR in 6.6% of patients. The median PFS and OS for the whole cohort of patients were 3.0 and 7.4 months, respectively.Optimal thresholds for NLR and PLR for all patients in the study were determined using ROC curves. For pre-NLR, the optimal cut-off value was 3.41 and the area under the curve (AUC) was 0.646 (95% CI: 0.551 – 0.741), and sensitivity and specificity were 0.682 and 0.417, respectively Figures . On the P = 0.007), high pre-PLR (P = 0.004), and post-NLR elevation (P = 0.010), yet negatively related to a rise in post-PLR (P = 0.579). No significant correlation between ORR and any of the indicators was observed. Findings from the analysis pre-NLR and pre-PLR in relation to treatment response are detailed in Tables Before treatment, 74 (48.7%) patients had a high NLR > 3.41 and 78 (51.3%) had a low NLR ≤ 3.41. In addition, 66 (43.4%) had a high PLR > 205.63, while 86 (56.6%) had a low PLR ≤ 205.63. After treatment, 72 (47.4%) patients experienced an elevation in NLR and 80 (52.6%) a drop in NLR. PLR increased in 57 (37.5%) patients and the reverse occurred in 95 (62.5%) patients. A low DCR was associated with a high pre-NLR . When PFS was evaluated according to the scoring system, the risk of death in patients with at least one risk factor was 1.85 times higher than that in patients without any risk factors , and the risk in patients with two risk factors was 3.21 times higher . When OS was assessed in a similar manner, patients with at least one risk factor were 1.77 time more at risk of death compared with patients with no risk factor . Patients with both risk factors had about 4.52 higher risk of death than those with no risk factor . There were differences in survival for the different scores  to 2 (extremely unfavorable). Differences in scores manifested different treatment responses and prognosis . Comparet scores . The pret scores .Our study showed a slightly lower treatment response and prognosis  when contrasted with the ALTER-0303 study. We speculate that the reasons for this discrepancy may be attributable to the differences in the study populations of the two. Patients in our study grossly speaking were with more advanced disease and worse ECOG scores, while using anlotinib as a relatively later line of therapy. The inclusion criteria of ALTER-0303 included stage IIIB patients and those using anlotinib as second-line therapy. Despite anlotinib exhibiting a small but indisputable benefit to survival in advanced NSCLC patients as third or further-line treatment, the absence of predictive factors necessitated a careful risk-benefit analysis before use in clinical practice. So, it is very important to identify appropriate patients according to the prognostic indexes so as to obtain an individualized treatment.Neutrophils secrete cytokines and chemokines, which promote tumor progression and induce resistance to cytotoxic drugs . FurtherIn conducting the study, we first systematically evaluated the predictive value of NLR, PLR, and their variations on third- or further-line anlotinib therapy for advanced NSCLC patients. Then, subsequently proved pre-PLR's value in predicting prognosis of patients on the regimen. The risk model detailed herein could be appropriate and useful in predicting treatment effect of anlotinib therapy as well as prognosis. It is important to note that our study was limited by it being a single center study with a relatively small sample size, hence with a minimal PR. Both factors introducing varying levels of bias. Since all patients were treated with anlotinib as at least third-line, it may have had some unknown impact on either subsequent therapy or on prognosis. Besides, a data-driven analysis often provides optimistic results. Consequently, validation analysis with independent data sets would be required in the future.Pre-PLR and post-NLR are generally inexpensive and easy-to-use hematological markers that have some relationship with treatment response and, per our study, were independent prognostic indicators for patients with advanced NSCLC receiving anlotinib as a third or further-line treatment. Notably, a low pre-PLR prior to anlotinib therapy could help in stratification patients on anlotinib therapy as it produced a considerable therapeutic benefit. At the same time, the patients whose risk value score was 0, as calculated from a combination of a low pre-PLR prior anlotinib therapy and the subsequent post-NLR, were shown to have superior effectiveness and better survival.


In vitro analysis of miR-29a effects on fibroblast and cardiac muscle cells pointed toward a protective role of miR-29a on aging-related fibrosis, by reducing cell migration and proliferation. In conclusion, our study indicates an adaptive increase of miR-29 in the natural aging process and suggests its role as a transcriptional repressor of SERPINH1, with a potential therapeutic value against adverse matrix remodeling and aging-associated tissue fibrosis.Deregulation of microRNA (miRNA) profile has been reportedly linked to the aging process, which is a dominant risk factor for many pathologies. Among the miRNAs with documented roles in aging-related cardiac diseases, miR-18a, -21a, -22, and -29a were mainly associated with hypertrophy and/or fibrosis; however, their relationship to aging was not fully addressed before. The purpose of this paper was to evaluate the variations in the expression levels of these miRNAs in the aging process. To this aim, multiple organs were harvested from young (2–3-months-old), old (16–18-months-old), and very old (24–25-months-old) mice, and the abundance of the miRNAs was evaluated by quantitative real-time (RT)-PCR. Our studies demonstrated that miR-21a, miR-22, and miR-29a were upregulated in the aged heart. Among them, miR-29a was highly expressed in many other organs, i.e., the brain, the skeletal muscle, the pancreas, and the kidney, and its expression was further upregulated during the natural aging process. Western blot, immunofluorescence, and xCELLigence analyses concurrently indicated that overexpression of miR-29a in the muscle cells decreased the collagen levels as well as cell migration and proliferation. Computational prediction analysis and overexpression studies identified SERPINH1, a specific chaperone of procollagens, as a potential miR-29a target. Corroborating to this, significantly downregulated SERPINH1 levels were found in the skeletal muscle, the heart, the brain, the kidney, and the pancreas harvested from very old animals, thereby indicating the role of the miR-29a-SERPINH1 axis in the aging process.  The persistent increase in human longevity has stimulated the interest of scientists in understanding the fundamental mechanisms underlying natural aging, which is the dominant risk factor for many pathologies, including cardiovascular diseases . Aging iDuring aging, the cardiac tissue architecture deteriorates, leading to a gradual decline in the cardiac reserves, a major pathophysiological feature of the heart in old individuals . CardiacMicroRNAs (miRNAs) are a class of small non-coding RNAs that regulate gene expression at the posttranscriptional level and together with other non-coding RNA molecules, are powerful regulators of a wide range of biological mechanisms in many disorders , 10. NotOne of the most influencing miRNAs in the cardiovascular system is miR-21, which is the major miRNA in cardiac fibroblasts, where it accounts for around 20% of total miRNAs . MiR-21 MiR-17-92 cluster, which consists of six mature miRNAs , has also been involved in cardiac aging. Within this cluster, miR-18a was reported to regulate the expression of several extracellular matrix proteins  and its Another miRNA involved in cardiac aging is miR-22. Previous studies reported a prohypertrophic effect of miR-22, with a critical role in cardiac remodeling –27. ThisAnother miRNA with a prominent role in cardiac hypertrophy and fibrosis is miR-29a, which is the most stable and most abundantly expressed member of its family . MiR-29 Aiming at identifying miRNAs with therapeutic value for the cardiac fibrosis associated with the natural aging process, we explored whether the expression of these four miRNAs changed during the natural aging process. Here, we report that miR-29a increase is a common feature of multiple organs in the natural aging process and it may represent a compensatory mechanism against cardiac fibrosis through SERPINH1 downregulation.n = 20), old , and very old .All animal experiments were conducted in accordance with the European Guidelines for Animal Welfare (Directive 2010/63/EU) and approved by the National Sanitary Veterinary and Food Safety Authority (nr 389/22.03.2018). C57Bl/6 mice were purchased from Jackson Laboratory and bred in the ICBP animal facility, under specific pathogen-free conditions, in a controlled environment of 12/12 h light/dark cycle, 21°C, 55–60% humidity, with chow and water ad libitum. Female and male C57BL/6J mouse cohorts used in this study were divided into three age groups: young , 0.1 mM norepinephrine, and 2mM L-glutamine and passaged every three days by 1:3 splitting onto gelatin/fibronectin-coated tissue flasks.The NIH/3T3 and HL-1 cell lines were purchased from ATCC and Merck Millipore, MA, USA, respectively. NIH/3T3 cells were cultured in high-glucose DMEM , supplemented with 10% fetal bovine serum (FBS)  and 1% antibiotic-antimycotic solution  and passaged two times a week at a density of 3 × 10g, 4°C for 5 min. In the end, all cellular pellets were pooled and resuspended in 10 ml of Dulbecco's Modified Eagle's Medium (DMEM)/F12 supplemented with 10% FBS, 1% antibiotic-antimycotic solution, 1 × L-glutamine , 10 ng/μl fibroblast growth factor (FGF) , and 10 μg/ml insulin for plating. Cells were allowed to adhere and proliferate in culture for 5 days before being used in experiments.Primary cardiac fibroblasts were isolated from mouse ventricles as previously described . BrieflyMice were anesthetized with ketamine: xylazine (100/10 mg/kg b.w.). The organs were harvested as follows: the cardiac ventricle, the mediastinal lymph nodes, the thymus, a portion of the right liver lobe, the spleen, the pancreas, the left kidney, the left quadriceps muscle, and the whole brain. The lymphatic organs were harvested only from young and old mice. The organs were immediately flash-frozen in liquid nitrogen in 2-ml RNase-free tubes and stored at −80°C. Organ powder was obtained from each collected organ by grinding in liquid nitrogen using a mortar and pestle and stored at −80°C until use.2O and stored at −80°C until use.The total RNA was extracted from 10 mg frozen organ powder in 1 ml of Trizol® reagent  with phenol/chloroform, by following the manufacturer's recommendations. For RNA isolation from cardiac fibroblasts, the miRNeasy kit , as per the protocol recommended by the manufacturer, was used. Total RNA was eluted in 30 μl of nuclease-free HFor quantification of mature miRNA strands, 25 ng of the total RNA were reverse-transcribed to complementary DNA (cDNA) using TaqMan® MiRNA assays and Applied Biosystems® TaqMan® MiRNA Reverse Transcription Kit. The reverse transcription was carried out on a Veriti™ 96-Well Thermal Cycler and the real-time quantitative reverse transcription PCR (RT-qPCR) was carried out using 0.2 μl primers and TaqMan probe, at a final reaction volume of 10 μl, on ViiA™ 7 RT-PCR System. The cycling conditions were as follows: enzyme activation at 95°C for 20 s, denaturation at 95°C for 1 s, and annealing/extension at 60°C for 20 s. Forty cycles of amplification were performed.T method and mouse S18 was used for normalization.The reverse-transcription reaction was made with 500 ng total RNA, using the High-Capacity cDNA Reverse Transcription Kit . Quantification of SERPINH1 was carried out using SYBR™ Select Master Mix  and ViiA™ 7 Real-Time PCR System. Relative expression was calculated using the comparative CCells were seeded onto a 24-well plate at 70% confluence, one day prior to transfection. Cells were transiently transfected with mature miR-29a-3p mimic  or scrambled miRNA control (negative control)  at a final concentration of 10 nM, using Lipofectamine™ RNAiMAX Transfection Reagent . The medium was replaced with a complete growth medium after 6 h of transfection, and cells were maintained in culture for another 24 h, before cellular assays.p-value was set as per the Benjamini-Hochberg method. The adjusted p < 0.01 was considered statistically significant.The miR-29a targets were predicted using three online analysis tools, such as TargetScan , DIANA-mFor PPI analysis, the target genes were mapped to the STRING v11 database  and onlyQuantification of total collagen level in the cardiac ventricle was performed using a Hydroxyproline Assay Kit  and following the manufacturer's recommendations. Briefly, 10 mg wet tissue was homogenized in 100 μl of water and hydrolyzed with 100 μl concentrated HCl for 3 h at 120 °C. The mixture was centrifuged and 30 μl supernatant was transferred into a 96-well plate and evaporated to dryness in a 60°C oven. The oxidation reaction was done using Chloramine T for 5 min at room temperature, and then the samples were mixed with DMAB/perchloric acid/isopropanol reagent and incubated for 90 min at 60°C. Hydroxyproline was measured spectrophotometrically at 560 nm using a standard curve.Cells were fixed in 4% formaldehyde, permeabilized with 0.1 % TritonX100, and incubated with anti-Collagen I  or anti-Collagen III  rabbit polyclonal antibody, overnight at 4 °C. Washed cells were then incubated with anti-rabbit IgG (whole molecule)-FITC secondary antibody  for 1 h at room temperature in the dark. Image acquisition was performed using a Leica DMi8 inverted fluorescent microscope  equipped with HC PL APO 20x/0.75 CS2 dry objective. Fluorophores were excited with a multi-LED Spectra-X light source  and images were captured with an sCMOS camera Leica DFC9000 and subsequently processed with Leica LAS X software. Z-stack images (15-μm depth) were acquired with LAS X Navigator module and mosaic merged and then digitally processed for extended depth of field. The fluorescence was quantified with the analysis tool of Las X software by averaging the least four random fields per each experimental condition and the results were expressed as mean fluorescence intensity.Cells were lysed in 2X Laemmli buffer containing proteases inhibitor cocktail , and protein concentration was determined using the Amido Black assay. A total of 40 μg of protein was loaded and resolved on 4–15% of precast polyacrylamide gel, followed by a transfer on 0.45 μm nitrocellulose membrane and Ponceau S reversible staining. After blocking 3 h in 5% skim milk in Tris-buffered saline containing 0.1% Tween 20 (TBS-T), the membranes were incubated with anti-collagen III antibody  overnight at 4°C. Membranes were rinsed in TBS and further incubated with HRP-conjugated secondary antibody  for 1 h. Protein bands were imaged after enhancement with a chemiluminescence agent  using an ImageQuant LAS4000 system . The protein expression was quantified by densitometry with TotalLab software and normalized to total protein as quantified by Ponceau S staining.3 cells/well) in a complete medium, and the cell index was recorded at 1-h interval for up to 48 h. The migratory capacity of the transfected cells was evaluated using CIM-plates, which are double-chamber systems (with 8-μm pore membranes) bearing microelectrodes onto the inner face of the membrane. Briefly, the cell suspension prepared in serum-free medium was added to the upper chamber (40 × 103 cells/well) and the cells were allowed to migrate through the membrane toward the lower chamber, containing the complete medium. When cells pass through the membrane in response to serum, they come in contact with the microelectrodes and generate impedance signals.The effect of miR-29a overexpression on HL-1 cell proliferation was evaluated with the xCELLigence System . This system monitors cellular events in real-time by using the microelectrodes integrated at the bottom of the specially designed E-plates. Briefly, 24 h after transfection initiation, the cells (expressing scramble miRNA sequence or miR-29a mimic) were seeded onto E-plates (15 × 105 cells was fixed with 70% ice-cold ethanol (3 h at −20°C) and then incubated with 20 μg/ml propidium iodide and 100 μg/ml RNAse A. The stained cells were analyzed with a CytoFlex flow cytometer . At least 20,000 events were analyzed per sample. The different phases of the cell cycle  were determined based on the fluorescent peak signal, which was proportional to cellular DNA content.Cell proliferation was analyzed using Propidium iodide staining and flow cytometry analysis. Briefly, the cell suspension containing 10P-value has been calculated using unpaired t-test for equal variances and unpaired t-test with Welch's correction for unequal variances. For cell cycle experiments, two-way ANOVA was used. Correlations were done using Pearson's correlation coefficient, for normally distributed data, or Spearman's correlation coefficient, for non-normally distributed data. A p-value less than 0.05 was considered statistically significant.All results were expressed as mean +/– SD. Statistical analysis was performed using GraphPad Prism 9.1.2 . To address whether the aging process impacts the abundance of these miRNAs in the myocardium, the expression levels were evaluated in the heart ventricle and the skeletal muscle of young (2–3-months-old) and old (16–18-months-old) animals . The ratGSE153214) nor qRT-PCR analysis identified any change in the expression level of this miRNA in isolated populations of cardiac fibroblasts and cardiomyocytes (data not shown). Interestingly, both miR-21a and miR-29a had significantly higher expressions in the heart draining lymph nodes in old animals, as well as in the spleen and the thymus , the spleen, and the thymus, knowing that the chronic low-grade systemic inflammation is a key pathological characteristic of aged myocardium. The qRT-PCR analysis showed no age-dependent alteration of miR-22 levels in the lymphoid organs in old animals , and gene thymus , which mTo further investigate whether the increases in miR-21a and miR-29a expression levels in the natural aging process are general aging features, several other organs have been included in the analysis. Having the steady or not-significantly increased expressions of miR-21a and miR-29a in the skeletal muscle of old animals (16–18-months-old) in comparison to young counterparts  were used for Gene Ontology (GO)-Biological Processes enrichment analysis . This anTo better understand the relationship between the predicted targets implicated in ECM organization, the PPI analysis was done using the STRING database. To visualize this network, the 46 genes included in this term were imported into Cytoscape. The generated network was further processed by eliminating the proteins with no direct interactions among them. The final network, containing 28 nodes and 188 interactions, is illustrated in To experimentally validate miR-29a as a modulator for SERPINH1 in the muscle cells, the gain-of-function approach was used in HL-1 cardiac muscle cells by transient transfection with miR-29a mimic, which resulted in a marked increase of the endogenous expression level of miR-29a, peaking at 1 day after oligonucleotide addition . The RT-in vivo, the transcriptional levels of SERPINH1 were measured in various organs from very old mice (24–25-months-old) relative to their young counterparts. Quantitative RT-PCR analysis revealed that SERPINH1 was decreased in all aged organs, except the liver (https://twc-stanford.shinyapps.io/maca/).To further validate the biological link between miR-29a and SERPINH1 synthesis he liver . Noteworhe liver . Our resTo further explore the function of miR-29a increase in the old heart and the skeletal muscle, the protein levels of collagen I and III, two targets of this miRNA which are both under the direct control of SERPINH1, were determined in miR-29a-overexpressing HL-1 cells. Immunofluorescence staining followed by fluorescence quantification showed decreased levels of collagen III  and collThe effects of miR-29a have been further evaluated in HL-1 cells by examining its effects on cell migration and proliferation. Cell migration was analyzed with the xCELLigence system, by following the capacity of cells to infiltrate the membrane pores of CIM plates in response to the chemoattractant molecules within the serum. The results showed a decreased migration ability of miR-29a overexpressing cells in comparison with the designed control cells . MoreoveIn conclusion, our data indicate that miR-29a is a potent posttranscriptional repressor of SERPINH1 in multiple organs and its upregulation is a common feature of the aging process, suggesting that it might represent a potential therapeutic target for age-related adverse matrix remodeling and tissue fibrosis.Deregulation of the miRNA profile in aging has been reported in several studies –42, yet It might be worth mentioning that, although the miR-18a level was not found significantly altered in the heart of old mice (16–18-months-old), its expression slightly, yet significantly, increased in the very old animals (data not shown), indicating that this miRNA gradually changed with aging, but was detected only at very old ages. Similar findings were reported by Schroen's group, which showed an increased expression of miR-18a in the heart of 104-week-old C57Bl/6 mice . This inBesides an increase in the size of the old ventricle, the miR-21 and miR-29a were also found in higher levels in other aged organs, including the skeletal muscle and the brain, which are in accordance with other previous studies –46. The via the TGF-β/Smad7 signaling pathway after myocardial infarction , is a molecular chaperone that belongs to the Serpin family, yet has no serin protease inhibitory effect. SERPINH1 is found primarily in the secretory pathway of collagen-producing cells, where it binds to and stabilizes procollagen, preventing incomplete folding and lateral aggregation prior secretion –70. Withvia luciferase reporter assay and western blot .ER-N, A-ML, MP, CM, and CN performed experiments. ER-N and AB designed the work, analyzed and interpreted the data, and wrote the manuscript. AB gave the final approval of the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by the Romanian Ministry of Education, PN-III-P4-ID-PCE-2020-1340-contract 122/2021.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.